Protocol 36655: Probe Assay - Ncapd3<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr9:27037322+27037410 89bp TAGCAGTTCAAAGGGTGAAGCA GCATGCCTGAATTGTTATTTC

Mut = 90 bp

Wt = 89bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

 

This mutation is a 494 bp deletion beginning at Chromosome 9 position 27,036,876 bp and ending after 27,037,369 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49346 GCA TGC CTG AAT TGT TAT TTC Common A
49347 TAG CAG TTC AAA GGG TGA AGC A Wild type Forward A
49348 TGG AGC CCT TGA GAC TAC AG Mutant Forward A
49349 Fluorophore-1 AAG GAA GAG CGT CAG ACA AAC CA Quencher-1 WT Probe
49350 Fluorophore-2 ACA GGT GTT TCC TTA ATC TTT ATG ACA GCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49346 0.40 uM
49347 0.40 uM
49348 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.